The present invention relates to recombinant adeno-associated virus (AAV) expression vectors and virions, as well as methods of using the same. More specifically, the present invention relates to recombinant AAV vectors and virions comprising genes encoding proteins deficient or lacking in lysosomal storage diseases, and methods of delivering recombinant AAV virions to a mammalian subject to treat lysosomal storage diseases.
Lysosomal storage diseases develop when cells are missing one or more of the many lysosomal enzymes essential for breaking down natural macromolecules. Typically, the undegraded molecules accumulate in the lysosomes to form storage vesicles, which eventually distort cellular structure and compromise function. The result is a chronic and progressive condition that causes a variety of physiological problems often leading to organ failure and premature death. Approximately 40 lysosomal storage diseases have been characterized, each of which involves deficiency in one or more specific enzymes.
A. The Mucopolysaccharidoses (MPS)
The Mucopolysaccharidoses (MPS) include seven subtypes. Each subtype is caused by a defect in an enzyme necessary for the sequential breakdown of glycosaminoglycans. The MPS diseases share many clinical features, although each type may vary in severity. Patients generally suffer from organomegaly, dysostosis multiplex, impaired hearing and vision, cardiovascular abnormalities and joint immobility. MPS I (Hurler""s Disease) is caused by a deficiency in xcex1-L-iduronidase necessary to breakdown the glycosaminoglycans, dermatan sulfate and heparin sulfate. MPS II (Hunter""s Syndrome) is due to the deficiency of iduronidase-2-sulfatase causing the accumulation of partially degraded heparan and dermatan sulfates. MPS III (Sanfilippo Syndrome) has four subtypes categorized by various deficiencies in sulpaminidase and N-acetylglucosamine 6-sulfatase. MPS IV (Morquoi Syndrome) is due to a deficiency of N-acetylgalactosamine-6-sulphate sulphatase. MPS V (Scheie Syndrome) has a deficiency in a-L-iduronidase that is qualitatively different from MPS I. MPS VI (Maroteaux-Lamy Syndrome) is due to deficiency of N-acetylgalactosamine-4-sulfatase. Finally, MPS VII (Sly Syndrome) is due to a deficiency in the lysosomal enzyme xcex2-glucuronidase (GUS) which aids in the breakdown of glycosaminoglycans (GAGs) (Sly et al. (1973) J Pediatr 82: 249-257).
Sly Syndrome is characterized by mental retardation, abnormal bone development, distorted features, and organ malfunctions leading eventually to organ failure (Neufeld et al. (1995) xe2x80x9cThe mucopolysaccharidoses.xe2x80x9d In: Scriver C R, Beaudet A L, Sly W S, Valle D (eds), The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill: New York, pp. 2465-2494). Although Sly syndrome is rare, a great deal is known about GUS and because there are animal models of this disease, Sly syndrome has become a paradigm for the study of lysosomal storage diseases in general and for gene therapy in particular.
Both dog (Haskins et al. (1984) Pediatr Res 18: 980-984) and mouse (Birkenmeier et al. (1989) J Clin Invest 83: 1258-1266) models of MPS VII have been studied. In these models, animals homozygous for mutations eliminating GUS activity display symptoms analogous to those in humans with Sly syndrome. In mice, the mutation is a spontaneous, single base pair deletion that results in a frameshift and a subsequent stop codon in the sequence coding for the GUS protein (Sands et al. (1993) Proc Natl Acad Sci USA 90: 6567-6571). Without GUS activity, GAGs cannot be completely catabolized and therefore accumulate in the lysosomes to form storage granules or vacuoles in almost all tissues. This in turn results in animals that have distorted facial features, defects in skeletal development, dwarfism, reduced learning. capacity, and early death at approximately 5 months of age (Birkenmeier et al. (1989) J Clin Invest 83: 1258-1266; Bastedo et al. (1994) J Clin Invest 94: 1180-1186). Another useful mutation in mice is the xe2x80x9cnearly nullxe2x80x9d GUS mutation, which was developed by V Chapman at Roswell Park Cancer Institute. These mice have only 1-3% of normal GUS activity yet do not display any overt MPS phenotypes. This implies that if a therapy provided even a minimal amount of GUS activity, the progression of MPS pathology would be halted or reversed.
Using the mouse model for MPS VII, several experimental therapies have been tried. These include enzyme replacement (Vogler et al. (1993) Pediatr Res 34: 837-840; O""Connor et al. (1998) J Clin Invest 101: 1394-1400), a variety of cell transplantation approaches (Bastedo et al. (1994) J Clin Invest 94: 1180-1186; Sands et al. (1993) Lab Invest 68: 676-686; Poorthuis et al. (1994) Pediatr Res 36: 187-193; Moullier et al. (1993) Nat Genet 4: 154-159; Naffakh et al. (1994) J Exp Clin Hematol 36: S11-S16; Wolfe et al. (1995) Gene Therapy 2: 70-78; Taylor et al. (1997) Nature Med 3: 771-774), and direct administration of adenoviral vectors (Li et al. (1995) Proc Natl Acad Sci USA 92: 7700-7704; Ohashi et al. (1997) Proc Natl Acad Sci USA 94: 1287-1292). All of these treatments provided measurable improvement, but none was entirely satisfactory.
Major problems that need to be overcome are the transient nature of enzyme replacement and adenoviral vector therapies, the invasiveness and potential complications of transplantation therapies, and the difficulty in restoring therapeutic GUS activity throughout the organism. The brain in particular has resisted most forms of therapy (Taylor et al. (1997) Nature Med 3: 771-774; Sly et al. (1997) Nature Med 3: 719-720).
B. Gaucher Disease
Gaucher Disease occurs in approximately 20,000 Americans. Many cases of mild disease are undiagnosed and the actual occurrence of the gene defect in the general population may be as high as 1 in 640.
There are three major types of Gaucher Disease, called Type 1, Type 2 and Type 3. Type 1, an adult-onset form, is the most common form and is non-neuropathic. The disease has a variable spectrum of severity. Clinical manifestations result from macrophages engorged with glucocerebroside which clog and enlarge the liver and spleen and displace normal bone marrow. This results in hepatosplenomegaly, bone pain and fractures, mild anemia and leukopenia, and bleeding due to displacement of platelet precursors in the bone marrow. Pulmonary infiltration by engorged macrophages can cause respiratory failure. Type 2 disease occurs in infants and is characterized by an acute neuropathic phenotype. This subtype has an extensive pathology, including the symptoms of Type 1, as well as oculomotor abnormalities, extreme retroflexion of the neck, limb rigidity, and seizures. Most infants with this form of Gaucher Disease die within the first 2 years of life. Type 3 disease is a juvenile onset form of the disease that presents with mild neurologic involvement in the first decade of life, progressing gradually toward severe neurologic impairment. The severity of Type 3 disease is intermediate to the other subtypes. In addition to the symptoms of Type 1, Type 3 pathology includes massive visceral involvement. CNS manifestations begin with disorders of eye movement, with progression to neurological symptoms equivalent to the severity of Type 2 disease.
A variety of mutations in the glucocerebrosidase (GC) gene cause Gaucher Disease. These gene defects include missense, frameshift and splicing mutations, deletions, gene conversions, and gene fusions with a pseudogene located 16 kb downstream of the GC gene. The most common mutation in Type 1 disease is an A to G transition at nucleotide position 1226 of the GC gene, causing an amino acid substitution which results in the non-neuropathic form of the disease. Neuropathic disease is associated with a T to C transition at nucleotide position 1448 of the GC gene.
Current treatment of Gaucher Disease includes hydration, analgesics and narcotics for pain during bone crises, in addition to the use of Vitamin D, calcium and bisphosphonate. Patients in bone crisis must also be monitored for osteomyelitis, which can be fatal. Joint replacement can be helpful in some patients. Splenectomy can alleviate thrombocytopenia and bleeding episodes, in addition to relieving symptoms due to physical pressure on other organs in the abdominal cavity.
Administration of GC can alleviate some symptoms. Patients receiving GC show improvement in anemia and thrombocytopenia after several months of therapy, followed by regression of organomegaly after about six months of therapy. Bone pain gradually decreases, but X-ray abnormalities persist. Enzyme therapy, however, does not improve CNS symptoms and can cost as much as $200,000 per year per patient. It is therefore cost-prohibitive for some patients. Moreover, the decision to treat a patient with GC is often based on the severity of disease and likely prognosis. Some patients are therefore denied treatment despite likely benefit. Additionally, treatment schedules minimize the effectiveness of enzyme treatment. GC has a half life of approximately 10 minutes and isolated enzyme administration creates a flood of GC during treatment sessions and a lack of GC between treatments. The short half life of GC also limits distribution. In an effort to reduce this bolus effect and decrease the cost of therapy, treatment regimens use less enzyme on a more frequent schedule. This regimen adds further disruption and inconvenience to patients. A continuous supply of GC by a patient""s own cells would eliminate enzyme half-life difficulties, remove any bolus effect, enhance distribution, resolve cost issues, and allow Gaucher""s patients to lead more normal lives.
C. Other Lysosomal Storage Diseases
Fabry""s disease is an X-linked lysosomal storage disease caused by an accumulation of glucosphingolipids in endothelial and perithelial smooth muscle cells. Patients lack xcex1-galactosidase A for cleaving the terminal xcex1-galactosyl of glycosphingolipids. Clinical manifestations of the disease include extremity pain, angiokeratomas of the skin and mucus membranes, corneal opacities, and renal impairment leading to proteinuria, lymphedema, uremia and hypertension. Death occurs from renal failure or heart disease.
Tay-Sachs disease is caused by accumulation of gangliosides in neuronal cells. Patients lack the hexaminidase A subunit necessary for formation of functional hexosaminidase heterodimer used to cleave hexose from gangliosides. The ganglioside accumulation in neuronal cells leads to a diffuse apoptotic cell death in the central nervous system. The disease is clinically variable with severe forms showing profound retardation and death by two to three years of age. Late-onset, chronic forms of the disease are dominated by neuronal symptoms such as progressive dystonia, spinocerebellar degeneration, motor neuron disease and psychosis.
Similarly, Sandhoff disease is a deficiency in hexosaminidase B, also interfering with formation of functional heterodimeric enzyme. Neurologic symptoms in Sandhoff disease are similar to Tay-Sachs, with additional symptoms related to visceral organ involvement.
Neimann-Pick Disease Types A and B result from accumulation of sphingomyelin due to lack of the lysosomal enzyme acid sphingomyelinase. Type A patients completely lack enzyme activity and, therefore, have more severe disease. Type A clinical pathology begins at 6 months of age with hepatosplenomegaly, lymphadenopathy, anemia and muscular weakness that affects the infants"" ability to feed. As macrophages filled with sphingomyelin infiltrate the lungs, bronchitis and pneumonia occur, with respiratory difficulties increasing throughout infancy. Type B patients possess residual enzyme activity, usually about 5 to 10 percent of normal. Clinical presentation is more variable in this form of the disease, with initial symptoms such as hepatosplenomegaly and pulmonary involvement occurring later in infancy or childhood. Type B patients do not typically have neurologic involvement. Type C Neimann-Pick Disease is characterized by accumulation of unesterified cholesterol due to a defect in intracellular trafficking of exogenous cholesterol. This form of the disease also presents with hepatosplenomegaly, progressive ataxia, dystonia, seizures and dementia. These patients present in late childhood and die in the second decade of life. There is currently no treatment for Neimann-Pick Disease.
Several Lysosomal Storage Diseases are caused by enzyme defects necessary for glycoprotein degradation. The glycoproteins are degraded in the lysosome by exoglycosidases, endo-xcex2-N-acetylglycosaminidase and aspartylglucosiminidase. Diseases include Mannosidoses, Fucosidoses, Sialodosis. Symptoms include skeletal deformities, mental retardation and visceromegaly. There is no current treatment for the glycoprotein-related Lysosomal Storage Diseases.
The Mucolipidoses are Lysosomal Storage Diseases characterized by defective transport of enzymes to the lysosome. Mannose-6-phosphate is the receptor ligand necessary for binding and uptake of enzymes into the lysosome. Lack of phospotransferases prevents addition of the mannose-6-phosphate to lysosomal enzymes, preventing the enzyme from entering the lysosome to carry out its function. Affected patients have cellular inclusion bodies filled with storage material and elevated levels of lysosomal enzymes in serum and body fluids. I-cell disease (Mucolipidoses II) and Pseudo-Hurler Polydystrophy (Mucolipidoses III) are the principal disorders. Pathology includes severe psychomotor retardation and premature death.
Acid Lipase Deficiency results in massive accumulation of cholesterol esters and triglycerides in most tissues of the body. There are two major phenotypes, Wolman Disease and Cholesterol Ester Storage Disease. Symptoms can include hepatosplenomegaly, steatorrhea, abdominal distension, gastrointestinal complications, adrenal calcification and extensive atherosclerosis. There is no specific therapy for acid lipase deficiency.
Sulfatide Lipidosis is a lysosomal storage disease caused by accumulation of cerebroside sulfate throughout the nervous system, the kidney and gallbladder. Patients with the disease are deficient in arylsulfatase A which catabolizes cerebroside sulfate in the lysosome. Clinical manifestations include gait disturbance, mental regression and urinary incontinence. As the disease progresses, blindness, loss of speech, quadriparesis, peripheral neuropathy and seizures develop. The two subtypes of the disease are Metachromatic Dystrophy and Multiple Sulfatase Deficiency. The only current treatment for these patients is bone marrow transplantation which merely slows the progression of the disease.
It is readily apparent that alternative and new therapies for the treatment of lysosomal disorders would be highly desirable.
Adeno-associated virus (AAV) provides a means for the delivery of genes to mutant cells. In a number of studies, the lacZ gene delivered by an AAV vector has been shown to make active xcex2-galactosidase in cultured cells and animal tissues (Kaplitt et al. (1994) Nat Genet 8: 148-154; Xiao et al. (1996) J Viral 70: 8098-8108; Kessler et al. (1996) Proc Natl Acad Sci USA 93: 14082-14087; Fisher et al. (1997) Nature Med 3: 306-312). In treated muscle of immunocompetent mice, this expression was shown to persist for 1.5 years (Xiao et al. (1996) J Viral 70: 8098-8108). Potentially therapeutic proteins that have been delivered and expressed by AAV vectors include erythropoietin (Kessler et al. (1996) Proc Natl Acad Sci USA 93: 14082-14087), cystic fibrosis transmembrane conductance regulator (Flotte et al. (1993) Proc Natl Acad Sci USA 90: 10613-10617), blood coagulation factor IX (Herzog et al. (1997) Proc Natl Acad Sci USA 94: 5804-5809; Monahan et al. (1998) Gene Therapy 5: 40-49), and GUS (Fisher et al. (1997) Nature Med 3:306-312. In none of these cases, however, were experiments designed to show that the AAV-mediated gene therapy actually affected the long-term course of a disease.
The present invention is based on the discovery that AAV-mediated delivery of genes encoding enzymes deficient or lacking in lysosomal storage disorders, results in sustained, long-term expression of the gene product in vivo. AAV virions can be administered via the bloodstream, directly to organs, such as the brain or liver, or intrathecally, such as through the spinal column, into cerebrospinal fluid.
In particular, results presented here show that subjects treated with AAV virions encoding xcex2-glucuronidase (QUS), acquire the ability to produce GUS for extended time periods. Furthermore, treatment has a beneficial effect on the subject, as it reduces or eliminates GAG storage in tissues. Morever, intrathecal injection of AAV virions into the cerebrospinal fluid to both neonatal and adult animals, results in therapeutic levels of GUS in the brain and the elimination of storage granules in brain tissue.
Similarly, delivery of AAV virions encoding glucocerebrosidase (GC), via the bloodstream, results in the production of GC in a variety of tissues, such as liver and spleen.
Accordingly, in one embodiment, the subject invention is directed to a method of delivering a recombinant AAV virion to a mammalian subject to treat a lysosomal storage disease. The method comprises:
(a) providing a recombinant AAV virion which comprises a nucleic acid molecule, the nucleic acid molecule comprising a gene encoding a protein defective or missing in the lysosomal storage disease operably linked to control elements capable of directing the in vivo transcription and translation of the gene; and
(b) delivering the recombinant AAV virion to the bloodstream of the subject, whereby the gene is expressed at a level which provides a therapeutic effect in the mammalian subject.
In certain embodiments, the recombinant AAV virion is delivered to the bloodstream by intravenous or intraarterial injection.
In yet further embodiments, the gene is expressed in the liver of the subject.
In another embodiment, the invention is directed to a method of delivering a gene encoding a protein defective or missing in a lysosomal storage disease to a mammalian subject. The method comprises:
(a) providing a recombinant AAV virion which comprises a nucleic acid molecule, the nucleic acid molecule comprising a gene encoding a protein defective or missing in the lysosomal storage disease operably linked to control elements capable of directing the in vivo transcription and translation of the gene; and
(b) delivering the recombinant AAV virion to the liver or brain of the subject, whereby the gene is expressed in the liver or brain at a level which provides a therapeutic effect in the mammalian subject.
In certain embodiments, the recombinant AAV virion is delivered to the brain by intrathecal injection.
In still a further embodiment, the invention is directed to a method of delivering a recombinant AAV virion to a mammalian subject to treat MPS VII. The method comprises:
(a) providing a recombinant AAV virion which comprises a nucleic acid molecule, the nucleic acid molecule comprising a gene encoding xcex2-glucuronidase operably linked to control elements capable of directing the in vivo transcription and translation of the gene; and
(b) delivering the recombinant AAV virion to the subject by intrathecal injection, whereby the gene is expressed at a level which provides a therapeutic effect in the mammalian subject.
In another embodiment, the invention is directed to a recombinant AAV vector comprising a gene encoding xcex2-glucuronidase operably linked to control elements capable of directing the in vivo transcription and translation of the gene in a mammalian cell.
In yet a further embodiment, the invention is directed to a recombinant AAV virion comprising a nucleic acid molecule that comprises a gene encoding xcex2-glucuronidase operably linked to control elements capable of directing the in vivo transcription and translation of the gene in a mammalian cell.
In another embodiment, the invention is directed to a method of producing a recombinant AAV virion. The method comprises:
(a) transfecting a host cell with (i) an AAV vector comprising a gene encoding xcex2-glucuronidase operably linked to control elements capable of directing the in vivo transcription and translation of the gene in a mammalian cell; (ii) AAV helper functions; and (iii) AAV accessory functions,
wherein the transfecting is done under conditions that allow the formation of a recombinant AAV virion; and
(b) purifying the recombinant AAV virion from the host cell.
These and other advantages of the present invention will become apparent upon reference to the accompanying drawing and upon reading the following detailed description.